Effect Of Clearing Phlegm And Removing Phlegm On The Temporal And Spatial Changes Of Estrogen-related Factors GPR30, Akt And P-Akt In AM Mice

Objective To investigate the mechanisms of adenomyosis by therapy of expelling dampness and dissipating blood stasis through observing the expression changes of GPR30、Akt、p-Akt in uterus tissues of AM mice before and after medication,and in different Course of disease which vary with estrus cycles.and study the distinction between ectopic and eutopic endometrium.Methods We firstly transplanted the pituitary gland of male mice into the uterine cavity of the female mice in order to construct the AM mice model.Then we divided these mice into seven groups randomly:high dose of Neiyikangfupian(expelling dampness and dissipating blood stasis)group(NYKFP group),medium dose of NYKFP group,low dose of NYKFP group;Gestrinonc group(GES group);Danefukangjiangao group(DEFKJG group);3 months group and 6 months group.At the same time,set the mice without operation or with fake operation as the control groups.Each group had 24 mice.Breed the mice for 3 months.Then two control groups and mice of 3 months model group were killed in estrum and interval period respectively.Then gave different drugs for another 3 months in each treatment group,and mice of 6 months group still fed by physiological saline for 3 months.Then we executed all the mice in the rest groups during in estrus and diestrus.We observed and analysed the endometrum glands and stroma invasion degree of muscular layer by pathological section and HE stain,and at the same time we surveyed the change of GPR30;Akt:p-Akt by the technique of immunohistochemisty.Results:1.We observed Ectopic tissues on muscular infiltration and evaluate the degree of adenomyosis severity.Compared with the normal group,the sham-operation group had no statistic significance(P>0.05),6 months group and 3 months group were both higher than normal group(P<0.01),and 6 months group was higher than 3 months group,but there was no statistical significance between the two groups(P>0.05).2.GPR30、Akt、p-Akt in the uterus endomembrane,the value of all the three had no statistic signification in normal group and sham-operation group(P>0.05).they gradually increased after the construction of mice model of AM,6 months group was higher than 3 months group(P<0.01),6 months group and 3 months group were notably higher than normal group and sham-operation group(P<0.01),Compared with 6 months group,the indicates were all regulated down after treatment,NYKFP high and medium dose groups、gestrinone group and DEFKJG group had notably statistic significance(P<0.01).NYKFP high dose group was lower than DEFKJG group and gestrinone group(P<0.01).The compare between different dozes of NYKFP had statistic significance different(P<0.01).3.Comparison of all of the groups in ectopic and eutopic endometrium:There was statistically significant between ectopic and eutopic endometrium in model groups about the relative expression of GPR30、Akt、p-Akt(P<0.01),and the eutopic endometrium was higher than normal uterus endomembrane(P<0.01),all the three were change with the estrus and diestrus cycle,and the estrus was higher than diestrus(P<0.01),except the relative expression of Akt in eutopic endometrium did not show the statistically significant between the estrus and diestrus cycle in the GES group,The rest of drugs did not have significant intervention effect on the periodic change.Conclusions:1.We observed Ectopic tissues on muscular infiltration and evaluate the degree score of adenomyosis severity,the degree showed that operation process of building had no effect to the molding,both groups molding success,and disease severity increases with the course of the disease.The molding method of male rat pituitary transfer in female rat uterine cavity in this study,which molding 3 months and 6 months the success rate are 100%,it is a worthy of promotion AM molding method.2.The occurrence of AM is related to the amount change of the correlation factors of estrogen effect GPR30 expression,and the expession of Akt and p-Akt which was the key factors of PI3K/Akt signal pathway.This experimental data show that the up-regulation of GPR30、Akt、p-Akt may lead the occurrence of AM.3.The therapy of expelling dampness and dissipating blood stasis could regulate down the correlation factors of estrogen effect GPR30 expression,and reduced the expession of Akt and p-Akt which was the key factors of PI3K/Akt signal pathway to restrain estrogen production to treat AM.But the regulation-mechanism of GPR30/PI3K/Akt pathway still need further study.4.NYKFP high and medium dose groups had a signification influence to the correlation factors of estrogen effect GPR30、Akt、p-Akt as well as DEFKJG group and gestrinone group,NYKFP high group was superior to the other each treatment group,the regulation degree of factors of estrogen effect related to the dose of NYKFP,high dose group>medium dose group>low dose group.So we can said that it is worth in-depth study of traditional Chinese medicine compound.5.The expression of GPR30、Akt、p-Akt were changed by estrous cycle,and the estrus was higher than diestrus.This will provide the oretical basis for AM to choose the best medication time.The therapy of expelling dampness and dissipating blood stasis did not have significant intervention effect on the periodic change.